EP0303925A1 - High repressible sequence for control of expression - Google Patents

Abstract

Expression control sequences for the expression of pro- and eukaryotic proteins, which have been obtained by combination of promoter sequences of low signal strength and high in vivo promoter strength and operator/repressor systems with high complex-formation rate, expression vectors which contain these expression control sequences, and microorganisms transformed with these expression vectors are described. Also described are methods for the preparation of pro- and eukaryotic proteins using these expression control sequences, expression vectors and transformed microorganisms. <IMAGE>

Description

The present invention relates to highly efficient and highly repressible expression control sequences, expression vectors which contain these expression control sequences, microorganisms transformed with these expression vectors and methods for their production by means of recombinant DNA technology. The present invention further relates to methods for the production of pro- and eukaryotic proteins by means of these highly repressible expression control sequences, expression vectors and transformed microorganisms.

The production amount of a protein in a host cell is determined by three main factors: the number of copies of its structural gene within the cell, the efficiency with which the structural gene copies are transcribed and the efficiency with which the resulting messenger RNA ("mRNA") is translated. The transcription and translation efficiencies are in turn dependent on nucleotide sequences which are normally located in front of the desired structural gene or the coding sequence. This nucleotide define sequences (expression control sequences), inter alia, that point to which the RNA polymerase attaches to initiate transcription (the promoter sequence) (see also The EMBO Journal 5,2995-3000 [1986]) and to which the ribosomes bind and with which mRNA (the transcription product) interact to initiate translation.

Not all expression control sequences are equally efficient. It is therefore often advantageous to separate the specific coding sequence for a desired protein from its neighboring nucleotide sequences and to link it with other expression control sequences in order to achieve a higher expression rate. After this is done, the recombined DNA fragment can be incorporated into a high copy number plasmid or bacteriophage derivative to increase structural gene copies within the cell, which can simultaneously improve the yield of desired protein.

Since the overproduction of a normally non-toxic gene product is often harmful to the host cells and lowers the stability of a special host cell vector system, an expression control sequence, in addition to improving the transcription and translation efficiency of a cloned gene, should be controllable in order to regulate expression during the To allow growth of the microorganisms. Examples of suitable expression control sequences are those which can be turned off during the growth of the host cells and which can then be turned on again at a desired time in order to promote the expression of large amounts of the desired protein by means of this expression control sequence.

Various expression control sequences that meet the above conditions have been used to express DNA sequences and genes encoding desired proteins. Such expression control sequences are known, for example, from Science 198, 1056-1063 (1977) (Jtakura et al.), Proc. Natl. Acad. Sci. USA 76: 106-110 (1979) (Goeddel et al.), Nature 283, 171-174 (1980) (Emtage et al), Science 205, 602-607 (1979) (Martial et al.), Gene 5, 59-76 (1979) (Bernard et al.), Gene 25, 167-178 (1983) (Ammann et al.), Proc. Natl. Acad. Sci. U.S.A., 80, 21-25 (1983) (de Boer et al.), As well as from European patent applications, publication no. 41767 and 186069.

According to the invention, it has now been found that by combining promoter sequences with low signal strength and high in vivo promoter strength with operator / repressor systems with a high complex formation rate (K _ass ), highly efficient and highly repressible expression control sequences are obtained. These expression control sequences are distinguished above all from the known ones by the fact that they can be repressed more than 1000 times and, after induction, mediate a high RNA synthesis rate (> 10 P _bla units) at ideal growth temperatures.

The present invention therefore relates to expression control sequences which are characterized by the combination of promoter sequences with low signal strength and high in vivo promoter strength with operator / repressor systems with a high rate of complex formation, in particular those which bring about a repression factor of> 1000.

The signal strengths of promoters are described by the complex formation rate (K _ass ) between promoter and RNA polymerase. The signal strengths of the promoter sequences of the expression control sequences according to the invention come K _ass values of about 1 · 10⁶-1.5 · 10⁸ M⁻¹ · sec⁻¹ in question, with a preferred K _ass value being about 6 · 10⁷ M⁻¹ · sec⁻¹. The K _ass values are determined as described in Example 3.

The in vivo promoter strength is defined by the RNA synthesis rate, which is triggered by a single promoter sequence, and is measured in P _bla units. The publication by Deuschle et al. (The EMBO Journal 5, 2987-2994 [1986]). RNA synthesis rates of 10-100, preferably more than 20 P _bla units are possible as high in vivo promoter strengths of the expression control sequences according to the invention.

The complex formation rate (K _ass ) of operator / repressor systems describes the speed at which a repressor binds to the operator. For the operator / repressor systems of the expression control sequences according to the invention, K _ass values of about 1 · 10⁸-1 · 10¹¹M⁻¹ · sec⁻¹ come into question, the K _ass value of 2 · 10⁹M⁻ described for the lac operator / repressor system ¹ · sec⁻¹ (Biochemistry 25, 3845-3852 [1986]) is particularly preferred.

The promoter sequences of the expression control sequences according to the invention are natural promoter sequences, as well as functional variants and combinations of these promoter sequences which have been specifically altered by mutation or synthesis, from gram-negative organisms such as E. coli, from gram-positive organisms such as B.subtilis and B.stearothermophilis , as well as from the corresponding phages. Preferred promoter sequences of the expression control sequences according to the invention are those from T-coliphages, the T7A1 promoter (the nucleotide sequence of which is shown in FIG. 8 and which is also referred to below as A1 promoter (P _A1 )) being particularly preferred.

Suitable operators / repressor systems are all systems which can be directly induced by chemical inductors and which, naturally or after corresponding changes (e.g. by mutation), enable a repression factor of> 1000, with these directly inducible systems not having an SOS function (lexA / recA system ) or temperature-inducible systems, such as the P _L operator / repressor system. Systems which can be regulated directly by chemical induction include, for example, the regulatory units of the lactose, galactose, tryptophan and tetracycline operons and other negatively controllable operons, ie operons regulated by operator / repressor action. For this, reference can be made to the textbook by Miller and Reznikoff ("The operon", Cold Spring Harbor Laboratory, 1980), as well as to the publication by Hillen et al. (J. Mol. Biol. 172, 185-201 [1084]). Particularly preferred operator / repressor systems are the natural lac operator / repressor system (Miller and Reznikoff, supra) as well as variants of these operator / repressor systems specifically modified by mutation or synthesis, which enable a repression factor of> 1000. The expression "repression factor" describes the quotient of the in vivo promoter strengths in the presence and absence of an inductor.

The expression control sequences according to the invention can be produced by known methods of recombinant DNA technology described in the literature. The textbook by Maniatis et al. ("Molecular Cloning" Cold Spring Harbor Laboratory, 1982).

The promoter sequences with low signal strength and high in vivo promoter strength can be combined with one or more operator / repressor system (s) to form an expression control sequence according to the invention. In the Using a single operator / repressor system, this can be located inside or outside the promoter sequence with low signal strength and high in vivo promoter strength. As a result, the operator / repressor system can be integrated into the promoter sequence, partially replace it or be preceded or readjusted at different intervals. An operator / repressor system is preferably integrated into the promoter sequence, a particularly preferred integration point being the spacer region between positions -12 and -29 (nomenclature as in FIG. 8). When using two operator / repressor systems, both can be located inside or outside or one inside and the other outside the promoter sequence with low signal strength and high in vivo promoter strength. Preferably, one is integrated in the spacer region and the other upstream in the 5′-direction ("upstream"), so that maximum cooperativity between the two operator sequences of the operator / repressor system is achieved via a repressor bond, as a result of which a repression factor of up to approximately 30,000 can be obtained.

The preferred expression control sequences of the present invention were obtained via chemical DNA synthesis, wherein functional parts of the lac operator sequence were combined with functional parts of the T7A1 promoter sequence. The construction of the preferred expression control sequences A1OPSA1, A10PSA1P21, A10PSA10P29, A1pOPSA1 and OPA1OPSA1 thus obtained is described in detail in Example 2 below. The nucleotide sequences of this expression control sequence are shown in FIG. 5 and FIG. 9 and the properties characterizing them are summarized in Table 8.

The lac operator sequences mentioned above are negatively regulated by the lac repressor. To get enough amounts of repressor molecules inside the cell ten, the corresponding gene can be expressed by known methods, such as for example by integrating the lacI ^q gene (Miller and Reznikoff, supra; Calos, Nature 274, 762-765 [1978]) into a vector or the chromosome of a bacterium in excess .

The expression control sequences according to the invention can be incorporated in a manner known per se into any suitable expression vector which can replicate in gram-negative and / or gram-positive bacteria. Suitable vectors can be composed of segments of chromosomal, non-chromosomal and synthetic DNA sequences, such as e.g. various known plasmids and phage DNA's. For this, reference can be made to the above-mentioned textbook by Maniatis et al. to get expelled. Particularly suitable vectors are plasmids of the pDS family (Bujard et al., Methods in Enzymology, ed. Wu and Grossmann, Academic Press, Inc., Vol. 155, 416-433 [1987]).

The expression control sequences according to the invention can, however, also be incorporated into the chromosome of gram-negative and gram-positive bacterial cells, as a result of which the selection agents necessary when working with vectors, such as e.g. Antibiotics, can be dispensed with.

Suitable DNA sequences which can be expressed by means of the expression control sequences according to the invention are those which encode pro- or eukaryotic proteins in vivo or in vitro. For example, such DNA sequences can encode enzymes, hormones, proteins with immunoregulatory, antiviral or antitumor activity, antibodies, antigens and other useful pro- or eukaryotic proteins.

The proteins that can be expressed by means of the expression control sequences according to the invention come for example malaria surface antigens, in particular the 5.1 surface antigen, the CS protein and the p190 protein from Plasmodium falciparum, lymphokines, interferons, insulin and insulin precursors, HIV 1 and 2 envelope and structural proteins, growth hormones and growth hormone-releasing factors in question.

• (a) Transformation eines geeigneten Bakteriums, vorteilhaft E.coli, Salmonella typhimurium oder B.subtilis mit einem Expressionsvektor in welchem die kodierende DNA eines gewünschten pro- oder eukaryoten Proteins mit einer vorstehend genannten Expressionskontrollsequenz operabel verknüpft ist;
• (b) Kultivierung des so erhaltenen Bakteriums unter geeig­neten Wachstumsbedingungen; und
• (c) Isolierung und, falls gewünscht, Reinigung des gewünsch­ten Proteins.

The methods for expressing DNA sequences coding for pro- or eukaryotic proteins by means of expression control sequences according to the invention built into expression vectors are known per se and are described in the above-mentioned textbook by Maniatis et al. described. They include the following measures:

• (a) transformation of a suitable bacterium, advantageously E. coli, Salmonella typhimurium or B. subtilis, with an expression vector in which the coding DNA of a desired pro- or eukaryotic protein is operably linked to an expression control sequence mentioned above;
• (b) culturing the bacterium thus obtained under suitable growth conditions; and
• (c) Isolation and, if desired, purification of the desired protein.

• (a) Einbau einer vorstehend genannten Expressionskon­trollsequenz, die mit der kodierenden Sequenz eines gewünschten pro- oder eukaryoten Protein operabel verknüpft ist, in das Chromosom eines geeigneten Bakteriums;
• (b) Kultivierung des so erhaltenen Bakteriums unter geeigneten Wachstumsbedingungen; und
• (c) Isolierung und, falls gewünscht, Reinigung des gewünschten Proteins.

The expression of DNA sequences coding for pro- or eukaryotic proteins by means of the expression control sequences according to the invention built into the chromosome of a bacterium, advantageously E. coli, Salmonella typhimurium or B. subtilis, can be carried out by the following method steps:

• (a) incorporation into the chromosome of a suitable bacterium of an expression control sequence mentioned above, which is operably linked to the coding sequence of a desired pro- or eukaryotic protein;
• (b) culturing the bacterium thus obtained under suitable growth conditions; and
• (c) Isolation and, if desired, purification of the desired protein.

The selection of a suitable host organism is determined by various factors known to the person skilled in the art. For example, compatibility with the selected vector, toxicity of the expression product, expression characteristics, necessary biological safety precautions and costs play a role and a balance must be found between all of these factors.

Suitable host organisms are gram-negative and gram-positive, for example E. coli, Salmonella typhimurium or B. subtilis strains. A particularly preferred host organism of the present invention is the E. coli strain M15. In addition to the above-mentioned E.coli strain, other generally available E.coli strains can also be used, for example E.coli 294 (ATCC No. 31446), E.coli RR1 (ATCC No. 31343) and E.coli W3110 (ATCC No. 27325) can be used.

The following examples help to better understand the present invention. These examples can be better understood when read in conjunction with the accompanying figures and tables. The following abbreviations and symbols appear in these figures and tables:
B, C, E, H, Ha, K, P, Sa, X and Xb denote interfaces for the restriction endonucleases BamHI, ClaI, EcoRI, HindIII, HpaI, KpnI, PstI, SalI <XhoI and XbaI, respectively.

repräsentiert die Promotoren der Gene bla, lacI und neo;

repräsentiert die ribosomalen Bindungsstellen der Gene bla, cat, neo, lacI und lacZ,

repräsentiert die Terminatoren t₀, T1 und TE, wobei ein Pfeil die funktio­nelle Orientierung der Terminatoren angibt;

reprä­sentiert die Expressionskontrollsequenzen A1OPSA1, lacOP29 und P_N25;

repräsentiert den Operator OP in der Expressionskontrollsequenz OPA1OPSA1; ⇆ repräsentiert die für die Replikation benötigte Region (repl.);

repräsentiert kodierende Regionen für Dihydrofolatreduktase (dhfr), Chloramphenicolacetyltransferase (cat), lac Repres­sor (lacI), β-Lactamase (bla), β-Galaktosidase (lacZ) und Neomycinphosphotransferase (neo).

represents the promoters of the bla, lacI and neo genes;

represents the ribosomal binding sites of the Genes bla, cat, neo, lacI and lacZ,

represents the terminators t₀, T1 and TE, with an arrow indicating the functional orientation of the terminators;

represents the expression control sequences A1OPSA1, lacOP29 and P _N25 ;

represents the operator OP in the expression control sequence OPA1OPSA1; ⇆ represents the region required for replication (repl.);

represents coding regions for dihydrofolate reductase (dhfr), chloramphenicol acetyl transferase (cat), lac repressor (lacI), β-lactamase (bla), β-galactosidase (lacZ) and neomycin phosphotransferase (neo).

illustration 1

Schematic representation and nucleotide sequence of the plasmid pDS1, tol⁺. The plasmid is shown schematically in Fig.1a. In the nucleotide sequence (Fig.1b), the recognition sites for the restriction endonucleases shown in the schematic representation are overlined, while the regions coding for β-lactamase (bla) or dihydrofolate reductase (dhfr) are underlined.

Figure 2

Schematic representation and nucleotide sequence of the plasmid pDS3. The plasmid is shown schematically in Fig. 2a. In the nucleotide sequence (Fig.2b) the recognition sites for the restriction endonucleases shown in the schematic representation are overlined, while the regions coding for β-lactamase (bla) or dihydrofolate reductase (dhfr) are underlined.

Figure 3

Schematic representation and nucleotide sequence of the plasmid pML3 / lacOP29. The plasmid is shown schematically in Fig.3a. In the nucleotide sequence (Fig. 3b), which is given as far as is known, the recognition sites for the restriction endonucleases shown in the schematic representation are overwritten, while those for β-lactamase (bla), lac repressor (lacI) or β-galactosidase ( lacZ) coding regions are underlined.

Figure 4

Schematic representation and nucleotide sequence of the plasmid pDMI, 1. The plasmid is shown schematically in Fig.4a. In the nucleotide sequence (Fig. 4b), the recognition sites for the restriction endonucleases shown in the schematic representation are overwritten, while the regions coding for neomycin phosphotransferase (neo) or lac repressor (lacI) are underlined.

Figure 5

Nucleotide sequences of the XhoI-EcoRI fragments with the expression control sequences lacOP29 (a), tacOP29 (b), N25OPSN25OP29 (c), A1OPSA1 (d), A1OPSCONA1 (e), A1pOPSA1 (f), OPUA1 (g), OPUA110CON (h) (i) or A10PSA10P29 (j). In these sequences, the nucleotide at which RNA synthesis starts (+1) and the regions at positions -10 and -35 are underlined, while lac operator sequences are overlined.

Figure 6

Nucleotide sequence of the EcoRI fragment with the promoter P _N25 . The nucleotide at which RNA synthesis starts and the regions at positions -10 and -35 are underlined in the sequence.

Figure 7

Nucleotide sequences of the XhoI-EcoRI fragments with the expression control sequences N25 * / 0 and N25OPSN25. The nucleotide at which RNA synthesis starts and the regions at positions -10 and -35 are underlined in the sequences, while lac operator sequences are underlined.

Figure 8

Nucleotide sequence of the XhoI-EcoRI fragment with the promoter P _A1 . The sequence underlines the nucleotide at which RNA synthesis starts (position +1) and the regions around positions -10 and -35.

Figure 9

Nucleotide sequence of the SalI-EcoRI fragment with the expression control sequence OPA1OPSA1. In the sequence, the nucleotide at which RNA synthesis starts and the regions around positions -10 and -35 are underlined, while lac operator sequences are overlined.

Figure 10

Schematic representation of the plasmid pDS1 / P _N25 , tol⁺. The P _N25 promoter is transcribed clockwise.

Figure 11

Schematic representation of the construction of the plasmid pDS3 / A1OPSA1. The A1OPSA1 expression control sequence is transcribed clockwise.

Figure 12

Schematic representation of the construction of the plasmid pDS3 / OPA1OPSA1. The nucleotide sequence of the SalI-EcoRI fragment with the expression control sequence OPA1OPSA1, from which it is transcribed clockwise, is shown in Fig.9.

Figure 13

Schematic representation of the construction of the plasmid pML3 / A1OPSA1. In this construction, the XhoI-EcoRI fragment of the plasmid pML3 / lacOP29 with the expression control sequence lacOP29 was replaced by the corresponding XhoI-EcoRI fragment with the expression control sequence A1OPSA1.

Figure 14

Schematic representation of the construction of the plasmid pML3 / OPA1OPSA1. Three DNA fragments were used to construct this plasmid: the SalI-EcoRI fragment from pDS3 / OPA1OPSA1 with the expression control sequence OPA1OPSA1, the EcoRI-ClaI fragment from pML3 / lacOP29 with parts of the lacZ gene and the larger ClaI-XhoI fragment from pML3 / lacOP29. In this construction, interfaces for SalI and XhoI were linked together, whereby both interfaces were destroyed.

Figure 15

Time-dependent course of the complex formation of RNAP and promoter P _N25 at a concentration of active RNAP of 0.42nM and a promoter concentration of 0.02nM.

Figure 16

Graphic representation of the expression -ln [(A₀-X) / A₀] as a function of the reaction time at a concentration of active RNAP of 0.42nM and a concentration of promoter P _N25 of 0.02nM.

Figure 17

Graphical representation of the time-dependent course of the complex formation of RNAP and promoter P _N25 at a promoter concentration of 0.02nM and a concentration of active RNAP of 0.32nM (test 2) and 0.15nM (test 3).

Figure 18

Graphical representation of the expression -ln [(A₀-X) / A₀] as a function of the reaction time at a promoter concentration of 0.02nM and a concentration of active RNAP of 0.32nM (experiment 2), or 0.15nM (experiment 3).

Figure 19

Determination of the complex formation rate for the promoter P _A1 with the promoter P _N25 as internal standard: -ln [(B₀-Y) / B₀] plotted against -ln [(A₀-X) / A₀].

Figure 20

Determination of the complex formation rate for the expression control sequence A1OPSA1 with the promoter P _A1 as internal standard: -ln [(B₀-Y) / B₀] plotted against -ln [(A₀-X) / A₀].

Figure 21

Determination of the factor for converting β-galactosidase units into P _bla units: β-galactosidase units under control of the expression control sequences tacOP29, N23OP29, OPUA1 and OPUA1CON plotted against the corresponding P _bla units.

example 1 Description of the plasmids used A. Principles

The plasmids pDS1, t₀1⁺ (Fig. 1), pDS3 (Fig. 2), pML3 / lacOP29 (Fig. 3) and pDMI, 1 (Fig. 4) were used to prepare and characterize the properties of the expression control sequences. E. coli cells transformed with these plasmids were found at the German Collection of Microorganisms (DSM) in Göttingen on December 11, 1984 [E. coli M15 (pDS1, t₀1⁺), DSM no. 3135], or on October 3, 1985 [E. coli M15 (pDS5 / RBSII, 3A + 5A; pDMI, 1), DSM no. 3517], or on August 5, 1987 [E. coli M15 (pDS3), DSM no. 4198; E. coli M15 (pML3 / lacOP29), DSM no. 4199], deposited under the Budapest Treaty.

B. The plasmid pDS1, t₀1⁺

The portion of pDS1, t₀1⁺ (Fig. 1) that lies between the interfaces for the restriction endonucleases XbaI and EcoRI and contains the replication region and the gene for β-lactamase, which confers ampicillin resistance on the cells, comes from the plasmid pBR322 (Bolivar et al., Gene 2, 95-113 [1977]; Sutcliffe, Cold Spring Harlor Symp. Quant. Biol. 43, 77-90 [1979]). The remaining part of the plasmid carries interfaces for the restriction endonucleases XhoI, EcoRI and BamHI followed by the gene of the dihydrofolate reductase of the mouse cell line AT-3000 (Chang et al., Nature 275, 617-624 [1978]; Masters et al., Gene 21, 59-63 [ 1983]), the terminator t₀ of the E. coli phage Lambda (Schwarz et al., Nature 272, 410-414 [1978]) and the promoter-free gene of chloramphenicol acetyltransferase (Marcoli et al., FEBS Letters, 110, 11 -14 [1980]).

C. The plasmid pDS3

The plasmid pDS3 (Fig. 2) differs from the plasmid pDS1, t₀1⁺ on the one hand in a region which, in addition to interfaces for various restriction endonucleases, the terminator T1 of the E. coli rrnB operon (Brosius et al., J. Mol. Biol. 148, 107-127 [1981]), and on the other hand due to the lack of the cleavage site for the restriction endonuclease EcoRI in the gene for chloramphenicol acetyltransferase.

D. The plasmid pML3 / lacOP29

The portion of pML3 / lacOP29 (FIG. 3) that lies between the interfaces for the restriction endonucleases SalI and EcoRI and contains the replication region and the gene for β-lactamase comes from the plasmid pBR322 (Bolivar et al., Supra; Sutcliffe, supra). The remaining part of the plasmid carries in addition to the complete lac I gene (Farabough, Nature 274.765 to 769 [1978]) encoding the lac repressor, the terminator T _E of the E. coli phage T7 (Dunn and Studier, J. Mol Biol., 166, 477-535 [1983]), the expression control sequence lacOP29 (see Example 2) and the promoter-free gene for β-galactosidase (Kalnins et al., EMBO J. 2, 593-597 [1983] ).

E. The plasmid pDMI, 1

The plasmid pDMI, 1 (Fig. 4) carries the gene of the neomycin phosphotransferase from the transposon Tn5 (Beck et al., Gene 19, 327-336 [1982]), which confers kanamycin resistance to E. coli cells and the lac I gene (Farabough, supra) with the promoter mutation I ^q (Calos, Nature 274, 762-765 [1978]), which encodes the lac repressor. In addition, the plasmid pDMI, 1 contains a region of the plasmid pACYC184 (Chang and Cohen, J. Bacteriol. 134, 1141-1156 [1978]), which contains all the information necessary for replication and stable transmission to the daughter cells. The plasmid pDMI, 1 is compatible with the plasmids described above and their derivatives.

Example 2 Production and cloning of the expression control sequences A. Principles

After the expression control sequences had been prepared, they were integrated into suitable plasmids to characterize their properties.

B. Production of Expression Control Sequences 1. Expression control sequences lacOP29, tacOP29, A1OPSCONA1, OPUA1, OPUA1CON, N25OPSN25OP29, P _A1 , A1OPSA1, A1pOPSA1, A10PSA10P21 and A10PSA10P29.

To produce these expression control sequences, single-stranded DNA fragments were first chemically synthesized (Bannwarth and Iaiza, DNA 5, 413-419 [1986]). These fragments were then hybridized and ligated to one another according to methods described in the literature (Maniatis et al., Supra). The sequences of the double-stranded XhoI-EcoRI fragments thus obtained with the corresponding expression control sequences are shown in Fig. 5 and Fig. 8.

2. Expression Control Sequences P _N25 , N25 * / 0 and N25OP29

The expression control sequences P _N25 , N25 * / 0 and N25OP29 (Fig. 6 and Fig. 7) can be prepared as described in the section above.

3. The expression control sequence OPA1OPSA1

The sequence of the expression control sequence OPA1OPSA1 is shown in Fig. 9. Their manufacture is described in Section D.

C. Integration of the Promoter P _N25  into the plasmid pDS1, t₀1⁺

The promoter P _N25 was integrated as part of an EcoRI fragment (see Fig. 6) into the plasmid pDS1, tRI1⁺ (Fig. 1) according to methods described in the literature (Maniatis et al., Supra), whereby the plasmid pDS1 / P _N25 , t₀1⁺ was obtained (Fig. 10). This plasmid was used as a source for obtaining the promoter P _N25 in larger quantities.

D. Integration of the expression control sequences into the plasmid pDS3

In a manner known from the literature (Maniatis et al., Supra), the expression control sequences lacOP29, tacOP29, OPUA1, OPUA1CON, P _N25 , N25 * / 0, N25OP29, N25OPSN25OP29, P _A1 , A1OPSA1, A10PSA10P21, A10PSA10P29, A1OPSCONA1 and A1pOPSA1 integrated into the plasmid pDS3 (Fig. 2). Such a cloning is shown schematically in Figure 11 using the example of the expression control sequence A1OPSA1.

The expression control sequence OPA1OPSA1 was produced by integrating a chemically synthesized DNA fragment, which contains a palindromic sequence of the lac operator, into the HpaI site of the plasmid pDS3 / A1OPSA1 (Fig. 11) (Fig. 12).

The pDS3 derivatives with the corresponding expression control sequences were used both to determine the complex formation rates between promoter and RNAP and to determine the promoter strengths of the individual expression control sequences.

E. Integration of the expression control sequences into the plasmid pML3 / lacOP29

The plasmid pML3 / lacOP29 (Fig. 3) carries the expression control sequence lacOP29 between the sites for the restriction endonucleases XhoI and EcoRI. According to methods described in the literature (Maniatis et al., Supra), derivatives of this plasmid were produced (FIG. 13), in which lacOP29 was replaced by one of the expression control sequences tacOP29, N25OP29, N25OPSN25OP29, A1OPSA1, A10PSA10P21, A10PSA10P29, A1OPSOPS1A1, A1p , or OPUA1CON has been replaced.

The plasmid pML3 / OPA1OPSA1 with the expression control sequence OPA1OPSA1 was prepared as described schematically in FIG. 14.

The pML3 derivatives obtained with the corresponding expression control sequences were used to determine the in vivo promoter strengths used under repressed conditions.

Example 3 Determination of the signal strength A. Principles

To determine the signal strength of the promoters or promoter / operator elements, the complex formation rate (K _ass ) between the E. coli RNA polymerase (RNAP) and the promoter P _{N25 was} determined absolutely. The K _ass values for the other signals were then determined using relative measurements using the P _N25 promoter as an internal standard.

B. Determination of K _ass  for RNAP and promoter P _N25

The K _ass for RNAP and promoter P _N25 was determined by means of filter binding experiments, with the aid of which the formation of RNAP / promoter complexes can be determined quantitatively as a function of the RNAP concentration and the reaction time. Since both RNAP and promoters are biological material, the evaluation of such experiments requires that the concentration of the active molecules is known for both reactants. For example, for the RNAP it is not the protein concentration that has to be taken into account but the concentration of the active polymerase molecules. The same applies to the promoter P _N25 and the single-stranded fd-DNA, which was used as a competitor for non-specific binding and for the termination of the association reactions. Accordingly, following the chapter on the theoretical derivation of the complex formation rates, the preparation and analysis of all reactants is discussed in detail before the actual attempts to determine K _{ass are} described.

1. Theoretical derivation of the K _ass

worin
R die freie Konzentration der RNAP,
P die freie Konzentration der Promotoren,
RP die Konzentration der RNAP/Promotor-Komplexe,
K_ass die Komplexbildungsrate, und
K_dissdie Dissoziationskonstante darstellt.The general reaction scheme applies to a second order bimolecular reaction between RNAP and promoters

wherein
R the free concentration of RNAP,
P the free concentration of the promoters,
RP the concentration of the RNAP / promoter complexes,
K _ace the rate of complex formation, and
K _{diss represents} the dissociation constant.

The following applies to the course of the reaction:

dP / dt = -K _ass x R x P + K _diss x RP

If the half-life of the RNAP / promoter complexes is long - i.e. the dissociation constant K _{diss is} small - the term K _diss x RP can be neglected and the following applies to the change in the promoter concentration over time:

dP / dt = -K _ass x R x P (1)

R and P change over time. However, if the RNAP is in a large excess, its concentration can be regarded as constant during the course of the reaction (ie if R = constant then dR / dt = O) and the following applies:

K _ass x R = m (2)

Accordingly, the following applies to the change in the promoter concentration over time (see equations (1) and (2)):

dP / dt = - mx P

or unshaped:

-dP / P = mx dt

After integrating this equation you get:

- lnP lmxt (3).

Equation (3) corresponds to a first-order reaction equation, ie formally the "decay of free promoter fragment into RNAP / promoter complexes" with the rate constant m [1 / sec]. P is the proportion of free promoter at time t (for t = O sec, P = 1). This proportion can be determined experimentally (see section 6). Knowing A₀ (total amount of promoters) and X (amount of RNAP / promoter complexes at reaction time t), the so-called "pseudo first order" constant m can be calculated using equation (3):

P = (A₀ - X) / A₀

and you get

m = - ln [(A₀ - X) / A₀] / t (4)

Since the formation of the promoter / operator complexes represents a bimolecular reaction of the second order, the constant m depends on the concentration of the RNAP. If this is known, the complex formation rate K _{ass can be} below Taking into account the equations

m = K _ass x R (2)

and

m = - ln [(A₀ - X) / A₀] / t (4)

can be calculated as follows:

K _ass x R = - ln [(A₀ - X) / A₀] / t

or reshaped:

K _ass = - ln [(A₀ - X) / A₀] / (tx R) (5)

When K _ass was derived, it was assumed that the reverse reaction, ie the decay of RNAP / promoter complexes, is negligible (K _diss x RP = O). Such decay is a first order reaction and is therefore independent of the concentration of the reactants. The association, however, depends on the concentration. Must therefore been chosen to be high for the determination of K _ass, the concentration of the reactants, the association process in a time that takes place, which can be in the decomposition of complexes neglected. This requires knowledge of the stability of the RNAP / promoter complexes.

Furthermore, it was assumed that the concentration of free RNAP can be regarded as constant during the course of the reaction. For this purpose, the RNAP must be in a large excess relative to the promoters, whereby it must be taken into account that the RNAP can also bind to non-specific DNA sequences. The number of such non-specific binding sites can be kept low by examining promoters on small, isolated DNA fragments will. With an excess of approximately 10 RAP molecules per promoter, the concentration of the free RNAP can be regarded as constant during the course of the reaction.

2. Preparation of the sample with the promoter P _N25

The plasmid pDS1 / P _N25 , t _o 1⁺ (Fig. 10) contains the promoter P _N25 on a 254 bp EcoRI fragment (Fig. 6). This plasmid was constructed by integrating the EcoRI fragment mentioned into the EcoRI site of the plasmid pDS1, t _o 1⁺ (FIG. 1), which is adjacent to the XhoI site. To purify the promoter fragment, the plasmid pDS1 / P _N25 , t _o 1⁺ was first purified (Maniatis et al., Supra) and 0.5 mg of this plasmid were then cut using the restriction endonuclease EcoRI according to the manufacturer's instructions. After phenol extraction and ethanol precipitation (Maniatis et al., Supra), the cut DNA was dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.6) and, after addition of sample buffer, electrophoresed in 6% polyacrylamide gels (Maniatis et al., supra). The fragments carrying the P _N25 promoter were then cut out of the gel with a scalpel and electrophoresed on DEAE paper (DE 81, Whatman, England). After washing three times with ethanol and TE buffer, the paper was air-dried, then the DNA was eluted with a buffer containing 10 mM Tris-HCl, 1 mM EDTA and 1.5 M NaCl, pH 7.6. After a 1: 3 dilution of the resulting DNA solution with TE buffer, the DNA was precipitated with ethanol, the sediment was washed with 80% ethanol and the DNA was then dissolved in TE buffer.

The concentration of this DNA solution was first as described by Mahler et al. described [J. Mol. Biol. 9, 801-811 (1964)], determined spectrophotometrically. Part of the DNA stock solution was then diluted 1:30 in TE buffer and the extinction was then compared to this dilution TE buffer measured as a blank. The following values were obtained:

ΔE₂₆₀ = 0.108 ΔE₂₆₀ / ΔE₂₈₀ = 1.86
ΔE₂₈₀ = 0.058

Taking into account the conversion factor of 1 ΔE₂₆₀ = 50 µg DNA / ml and the dilution factor, there was a DNA concentration of 162 µg / ml for the stock solution. Taking into account the length of the promoter fragment (254 bp), this value corresponds to a concentration of 0.96 pmol DNA fragment / µl. The ratio of the absorbances at 260 and 280 nm allows statements about the purity of the DNA solution. The value for ΔE₂₆₀ / ΔE₂₈₀ of 1.86 indicates a high purity of the DNA solution.

The concentration of the DNA solution was additionally determined by comparing this solution with an RNA-free solution of the plasmid pDS1, t _o 1⁺ of known concentration (ΔE measurement). For this purpose, 0.09 pmol of the DNA solution containing the promoter fragment was mixed with 0.2, 0.1, 0.5 and 0.025 pmol of the cut pDS1, t _o 1⁺ plasmid DNA and characterized in a 6% PAA gel (Maniatis et al. , supra). The pherogram obtained was evaluated densitometrically, a concentration of 0.8 pmol / μl being obtained for the promoter fragment compared to the cut pDS1, t _o 1⁺ plasmid DNA.

To determine the purity of the promoter fragment, 0.36 pmol of this fragment was first characterized in a PAA gel. No contamination could be detected after staining with ethidium bromide. Furthermore, the fragment was radioactively labeled with 32 P (see below) and also characterized electrophoretically. After autoradiography, only the promoter fragment could be detected.

In summary, it can be stated that the isolated EcoRI fragment with the promoter P _{N25 was} not contaminated with substantial amounts of RNA or DNA and was present in a concentration of 0.88 ± 0.08 pmol / µl. In the experiments for determining the concentration of the active polymerase (see below), a mixture of the isolated EcoRI fragment with the promoter P _N25 and of the corresponding fragment radioactively labeled with 32 P was used as the promoter sample. This radioactively labeled DNA was produced as follows: First, the DNA was dephosphorylated using CIP (calf intestinal alkaline phosphatase) (Maniatis et al., Supra) and then using T4 polynucleotide kinase by incorporating 32 P (γ-32 P ATP, Amersham, 3000 Ci / mmol) labeled (Maniatis et al., Supra). The radioactively labeled EcoRI fragment was then purified with the promoter P _N25 by chromatography on Sephadex G75 (Pharmacia, Sweden). To determine the specific activity of this sample, the radioactivity was measured on the one hand (Cerenkow) and on the other hand the DNA concentration was determined electrophoretically against the pretreated, isolated EcoRI fragment (see above). By mixing the untreated and the radioactively labeled EcoRI fragment, samples with the promoter P _N25 of defined concentration and specific activity were finally obtained.

3. Preparation and characterization of single-stranded M13mp8 DNA

Single-stranded M13mp8 DNA was used both as a competitor for the non-specific binding of RNAP to DNA and for the termination of association reactions. The preparation and characterization of this DNA is described below. E.coli JM 101 cells (Maniatis et al., Supra; GIBCO-BRL, Basel) were mixed with 0.01 pmol M13mp8 RFI-DNA (Pharmacia, Sweden) according to that described by Morrison Method [Methods of Enzymology 68, 326-331 (1979)]. The cells were then plated on indicator plates containing isopropylthiogalactoside (IPTG) and X-gel (5-bromo-4-chloro-3-indolyl-β-D-galactoside). For this, reference can be made to the textbook by JH Miller "Experiments in Molecular Genetics", Cold Spring Harbor Laboratory (1972)] and the article by Measurement and Vieira in Gene 19, 269-276 (1982). Cloudy plaques with transformed E.coli JM 101 cells were cut out of the top agar layer of these indicator plates with a Pasteur pipette, transferred to 10 ml LB medium and incubated for 8 hours in a shaking incubator (37 ° C., 220 rpm). The cells were then centrifuged off and the supernatant containing M13 mp8 phages was used for the new infection of E. coli JM 101 cells. For this purpose, E.coli JM 101 cells were first grown in 500 ml of M9 minimal medium (JH Miller, supra) to an OD 2 = 2 (37 ° C; 220 rpm) and then the supernatant containing 1 ml of M13mp8 phage (see above) added to these cells. After a further 12 hours of incubation at 37 ° C., the cells were centrifuged off. The phages were precipitated from the supernatant with 3% polyethylene glycol (PEG-6000) and 0.5 M NaCl and then centrifuged off. The sediment was taken up in 20 ml of TE buffer and extracted twice at 65 ° C. with phenol (equilibrated in 200 mM Tris HCl, pH 8) and phenol / chloroform (1: 1).

The single-stranded M13 mp8 DNA thus released was precipitated with ethanol and dissolved in 1 ml of TE buffer. The concentration of this DNA solution was determined spectrophotometrically (Mahler et al., Supra), based on the conversion factor of 1 OD₂₆₀ = 36 µg single-stranded DNA / ml.

4. Determination of the concentration of active polymerase in RNAP solutions

To determine the concentration of active E. coli RNA polymerase in RNAP solutions, after incubating the RNAP in the presence of an excess of promoter fragments, the proportion of the RNAP / promoter complexes formed was determined with the aid of filter binding experiments. The concentration of the bound E. coli RNA polymerase was then calculated from this fraction, taking into account the amount of promoter fragments used. The concentration of bindable RNAP molecules determined in this way was equated with the concentration of the free, active RNAP.

Such a concentration determination is described below. 0.12 pmol promoter fragment (see Section B, 2 .; specific activity: 4 x 10⁴ cpm / pmol) were for 2 minutes at 37 ° C in 50 ul binding buffer (BP) (20 mM Tris-HCl, pH 8.0 10 mM MgCl₂ , 0.1 mM EDTA, 1 mM DTT, 5% glycerin, 120 mM KCl). E.coli RNAP (Pharmacia, Sweden) was diluted at 0 ° C in BP containing 120 mM KCl. Each 100 ul of such a dilution was incubated for 2 minutes at 37 ° C and then pipetted to the solution with the promoter fragment. The batches were kept at 37 ° C. for 5 minutes, after which 300 μl BP containing 0.8 μg single-stranded M13mp8 DNA (see Section B, 3.), which had been preincubated at 37 ° C. for 2 minutes, were added. After a further 5 minutes of incubation at 37 ° C., the batches were filtered at 37 ° C. over nitrocellulose. Such a filtration process is described below. First, a nitrocellulose filter (nitrocellulose filter BA, 0.45 µm; Sartorius, Göttingen) was cut into small square pieces (4 x 4 mm) and soaked in BP containing 40 mM KCl. Such a piece was then transferred to a likewise soaked glass fiber filter (GF / A, Whatman), which is on a There was a glass frit. This glass frit was again in a 37 ° C water bath and was connected to a water jet pump. The reaction batches (see above) were filtered at a filtration rate of 1 ml / minute. The filter was then rinsed with 1 ml BP containing 40 mM KCl (preheated to 37 ° C). To elute the filter-bound DNA, the nitrocellulose filter was crushed with a pipette tip (Eppendorf) in an Eppendorf tube and, after adding 20 μl elution buffer (EP) (10 mM Tris / HCl, 1 mM EDTA, 0.1% SDS, pH 8), squeezed out with a pipette tip . The elution batch was placed on ice for 30 minutes and then centrifuged for 5 minutes (Eppendorf benchtop centrifuge, 12000 rpm). The elution solution was then removed and transferred to another Eppendorf tube. 50 μl of TE buffer were added to the filter and the mixture was shaken for 3 minutes (Eppendorf shaker). The rinsing solution was then lifted off and combined with the elution solution. After a total of three elution and rinsing steps, about 95% of the bound DNA was eluted (control by counting the residual activity bound to the filter; Cerenkow). After removing any existing filter fragments by centrifugation (see above), the radioactivity of the eluate (210 µl) was measured.

The calculation of the concentration of active polymerase in an RNAP solution is described below. If there is an excess of active polymerase compared to the promoter fragment, the count values form a plateau, the plateau value corresponding to an amount of 0.12 pmoles of promoter fragment (same fragment concentration as used in the experiments). From the count values obtained in the deficit of polymerase compared to the promoter fragment, the concentration of active polymerase can now be determined for each RNAP dilution, taking into account the dilution factor, using the ratio of the deficit RNAP count / excess RNAP count.

5. Determination of the half-life of RNAP / promoter complexes

When deriving the K _ass , it was assumed that the decay of RNAP / promoter complexes is negligible during the test period (see Section B, 1.). This assumption was verified in experiments that were used to determine the half-life of RNAP / promoter complexes. Such a determination is described below. First, RNAP / P _N25 complexes were incubated by incubating 0.06 pmoles of promoter fragment (see Section B, 2 .; specific activity: 2.4 × 10⁶ cpm / pmole) with 1.2 pmoles of active polymerase in BP containing 120 mM KCl for 5 minutes at 37 ° C. educated. After addition of 5 μg of single-stranded M13mp8 DNA (see Section B, 3.), samples were taken from the mixture, which was kept at 37 ° C., at various times (0-180 minutes) and filtered at 37 ° C. through nitrocellulose filters (see section B, 4.). The filter bound radioactivity was measured as measured in Section B, 4. and the values obtained plotted against the reaction time. The evaluation of this diagram showed that under these experimental conditions, complexes from RNAP and promoter P _{N25 have} a half-life of approximately 3 hours.

6. Determination of the K _ass  for RNAP and the promoter P _N25

The kinetic measurements for determining K _ass for RNAP and the promoter P _N25 were carried out under "pseudo first order" conditions, ie with a high RNAP excess. For this purpose, the test conditions and reaction times are chosen so that the back reaction, ie the chance of RNAP / promoter complexes formed, can be neglected (test duration maximum 7 minutes with a half-life of the complexes of approximately 180 minutes; see Section B, 5.). The binding reactions were all carried out according to a uniform scheme, with three independent test series, both the reaction volumes and the concentration of the reactants were varied. The P _N25 promoter fragments were preincubated in buffer before the RNAP solution, likewise preincubated in buffer, the concentration of active polymerase of which was determined in experiments carried out in parallel as described in Section B, 4., was added and the reaction was started by mixing the mixture has been. After the selected reaction times (1-120 seconds) the reaction was stopped by adding single-stranded M13mp8 DNA (see Section B, 3.). The RNAP / promoter complexes formed were then quantified using filter binding experiments (see Section B, 4.). Three attempts to determine the complex formation rates K _{ass are} described below.

Trial 1

50 ul P _N25 promoter fragment (0.045 nM, specific activity 3 x 10⁶ cpm / pMol) in BP containing 120 mM KCl were incubated at 37 ° C for 2 minutes. RNAP was gradually diluted at 0 ° C (1:10 to 1:20 in each case) in BP containing 120 mM KCl. Then 100 µl of a 1: 5000 dilution were preincubated for 2 minutes at 37 ° C (the concentration of active polymerase was 0.42 nM; see Section B, 4.). The solutions with the P _N25 promoter fragment and the RNAP were combined and the association reaction started by mixing the mixture. After 10 seconds of incubation at 37 ° C., 300 μl of BP solution incubated at 37 ° C. for 2 minutes, containing 0.8 μg of single-stranded M13mp8 DNA, were added. After incubation for 5 minutes at 37 ° C., the batch was filtered over nitrocellulose at 37 ° C., the filter was rinsed with 200 μl BP containing 40 mM KCl and the filter-bound radioactivity was then determined (Cerenkow). Another eleven experiments were carried out under the same conditions, the association reactions after 1, 2, 3, 4, 5, 6, 7, 8, 15, 20, 60 and 120 seconds were stopped by adding the single-stranded M13mp8 DNA. The count values obtained from all twelve partial experiments were plotted against the reaction time in Figure 15, the dashed line representing the maximum filter-bindable radioactivity. To determine this value, 50 ul P _N25 promoter fragment with 100 ul BP containing 120 mM KCl and 1 pmol of active polymerase were incubated at 37 ° C for 3 minutes. The reaction was then stopped and the mixture was worked up as described. The maximum bindable radioactivity (A₀) corresponds to the amount of promoter fragments used (or 100% RNAP / P _N25 complexes). The difference between A₀ and the radioactivity (X) bound at time t is a relative measure of the free promoter fragment. The expression (A₀-X) / A₀ then indicates the proportion of free promoter fragment at time t. The expression -ln [(A₀-X) / A₀] was calculated for all partial tests and plotted against the reaction time t (see Fig. 16). The slope m of the regression line corresponds to the rate constant m of equations (3) and (4) from section 1 and was calculated as m = 0.12 / second. Taking into account the concentration of active polymerase of 0.42 nM (as determined in Section B, 4.), the relationship K _ass = m / R (equation (2) in Section 1) resulted in a K _ass of 2.8 x 10⁸ M⁻¹ · Sek⁻¹.

Trials 2 and 3

Experiments 2 and 3 were carried out in an analogous manner to experiment 1, the reactants being present in the following concentrations, based on the reaction solutions:

Trial 2

Promoter fragment P _N25 : 0.02 nM
active RNAP: 0.32 nM
Response times: 2, 4, 8, 10, 15, 20 and 60 seconds

Trial 3

Promoter fragment P _N25 : 0.02 nM
active RNAP: 0.15 nM
Response times: 2, 4, 8, 10, 15, 20 and 60 seconds

The count values obtained were evaluated as described for experiment 1 (see FIGS. 17 and 18), the following values being determined:

Trial 2

m = 0.104 / sec; K _ass = 3.2 x 10⁸ M⁻¹ · sec⁻¹

Trial 3

m = 0.04 / sec .; K _ass = 2.7 x 10⁸ M⁻¹ · sec⁻¹

Averaged over all three experiments, the following value was obtained as the complex formation rate K _ass for RNAP and the promoter P _N25 :

K _ass = 2.9 x 10⁸ M⁻¹ · sec⁻¹.

This value can be associated with an error of approximately 15%, since the measurement errors in the concentration determination of promoter fragments and selective RNAP as well as in the filter binding tests are of the order of magnitude of 10%.

C. Determination of the complex formation rates for RNAP and various expression control sequences via relative measurements with the promoters P _N25  or P _A1  as an internal standard 1. Theory

The complex formation rates for RNAP and the expression control sequences tacOP29, N25 * / 0, N25OP29, N25OpSN25Op29, A1, A1OPSA1, A1OPSCONA1, A1pOPSA1 and OPA1OPSA1 were determined via relative measurements with the promoters P _N25 and P _A1 as internal standards. The following considerations were used:

In a mixture of different promoters, these compete for RNAP according to their complex formation rates, if this is in deficit.

Second for the temporal change of the concentration of promoter of a bimolecular reaction order between RNAP and promoter applies at high half-life of the complexes formed equation (1) (see section B) dP / dt = -K _ass x R x P.

In a reaction mixture with different promoters, the concentration of free RNAP is the same for all promoters at all times. The following relationships therefore apply to two promoters a and b with the complex formation rates K _{ass, a} and K _{ass, b} :

- dP _a / P _a x 1 / K _{ass, a} = R x dt
- dP _b / P _b x 1 / K _{ass, b} = R x dt

Since R x dt is the same for both promoters, the following applies:
- dP _a / P _a x 1 / K _{ass, a} = - dP _b / P _b x 1 / K _{ass, b}

or reshaped:

- dP _a / P _a = - K _{ass, a} / K _{ass, b} x dP _b / P _b

After integration you get:

- ln [(A₀-x) / A₀] = - K _{ass, a} / K _{ass, b} x ln [(B₀-y) / B₀]

respectively.

K _{ass, b} = K _{ass, a} x -ln [(B₀-y) / B₀] / - ln [(A₀-x) / A₀] (6)

with A₀ and B₀ as the total amount of the promoters a and b bound in the RNAP excess, and with x and y as the amount of complexes formed from RNAP and promoter a in the RNAP deficit, or from RNAP and promoter b.

2. Determination of complex formation rates with the promoter P _N25  as an internal standard

Radioactive-labeled DNA fragments (see section B.2) with the corresponding expression control sequences (see example 2) were mixed in parallel in a volume of 30 μl with different amounts of RNAP for 2 min at 37 ° C. in binding buffer (see section B. 6) incubated.

Then 20µl binding buffer (37 ° C) with 1µg single-stranded M13mp8-DNA (see section B.3) was added to terminate the association reaction before after another 2 min at 37 ° C the batches were filtered through nitrocellulose filters (see section B.4 ).

The complexes retained on the filter were eluted (see Section B.4) and, after extraction with phenol, precipitated with ethanol before the DNA was taken up in 30 µl sample buffer. Subsequently, a third of the existing DNA was electrophoresed in 6% polyacrylamide gels with 8.3 M urea according to the instructions of Maniatis (supra) and visualized by autoradiography.

For each ratio of RNAP to promoter, the amount of bound promoter (X for P _N25 , Y for the promoter to be measured) was first determined for each promoter. To the individual tracks in the autoradiograms, which each corresponded to a test with a certain amount of RNAP, were measured densitometrically (densitometer Elscript 400, from Hirschmann, Unterhachingen, FRG). The mean values for the total amounts of bindable promoters (A₀ for P _N25 , B₀ for the promoter to be measured) result from traces whose ratio of RNAP to promoter was greater than / equal to 1.

To determine the complex formation rates, the values for -ln ((A₀-x) / A₀) and -ln ((B₀-y) / B₀) were calculated for each RNAP / promoter ratio and plotted against each other. The slope of the straight line m obtained corresponds to the expression -ln [(B₀-y) / B₀] / - ln [(A₀-x) / A₀] from equation (6). With the aid of this value and the complex formation rate of the promoter P _N25 , the complex formation rate for the measured promoter is now obtained using equation (6).

The determination of the complex formation rate for promoter A1 is described below as an example. Approximately 0.02 pmol fragment with the promoter P _N25 (∼10,000 cpm) and approximately 0.02 pmole fragment with the promoter P _A1 (∼10,000 cpm) with different amounts of RNAP (0.004-0.12 pmol) were incubated in seven parallel batches. Table 1 shows the RNAP / promoter ratios, the densitometric values obtained and the values calculated therefrom for the individual experiments. Table 1 RNAP / promoter densitrometric value -ln [(A₀-x) / A₀] -ln [(B₀-y) / B₀] P _N25 P _A1 3rd 25338 ^a 26844 ^a 1.5 30331 ^a 30510 ^a 1.0 25361 ^a 22977 ^a 0.5 23085 (x₁) 17586 (y₁) 1.97 1.05 0.3 15729 (x₂) 10843 (y₂) 0.89 0.51 0.2 13226 (x₃) 9296 (y₃) 0.67 0.41 0.1 7203 (x₄) 3535 (y₄) 0.31 0.14 ^a Values for determining the mean value A₀ or B₀ for the promoter P _N25 or P _A1 .

Figure 19 shows the values listed in Table 1 graphically. The value m = 0.5 was obtained for the slope of the straight line. With the help of equation (6) (see above): K _{ass, A1} = K _{ass, N25} xm (K _{ass, N25} = 2.9x10⁸M⁻¹xsec⁻¹) K _{ass, A1} = 2.9x0.5 x 10⁸M⁻¹xsec⁻¹ K _{ass, A1} = 1.5x10⁸M⁻¹xsec⁻¹

The following complex formation rates were obtained for the other expression control sequences.
tacOP29: K _ass = 0.85x10⁸M⁻¹xsec⁻¹
N25 * / 0: K _ass = 2.9x10⁸M⁻¹xsec⁻¹
B25OP29 K _ass = 2.9x10⁸M⁻¹xsec⁻¹
N25OPSN25OP29: K _ass = 2.9x10⁸M⁻¹xsec⁻¹

These values can have an error of approximately 15%.

3. Determination of the complex formation rates with the promoter P _A1  as an internal standard

The complex formation rates of the expression control sequences A1OPSA1, A1OPSCONA1, A1pOPSA1 and OPA1OPSA1 were determined with the promoter P _A1 as an internal standard as described in section C.2. The determination of the complex formation rate for the element A1OPSA1 is described below as an example. Table 2 shows the RNAP / promoter ratios, the densitometric values obtained and the values calculated for the four parallel experiments with different amounts of RNAP: Table 2 RNAP / promoter densitometric value -ln [(A₀-x) / A₀] -ln [(B₀-y) / B₀] P _A1 A1OPSA1 3rd 61856 (A₀) 69018 (B₀) 0.00 0.00 0.5 41477 (x₁) 24983 (y₁) 1.11 0.45 0.2 22484 (x₂) 9054 (y₂) 0.45 0.14 0.1 11303 (x₃) 5517 (y₃) 0.20 0.08

Figure 20 shows the values listed in Table 2 graphically. The value m = 0.4 was obtained for the slope of the straight line. With the help of equation (6):

K _{ass, A1OPSA1} = K _{ass, A1} xm (K _{ass, A1} = 1.5x10⁸M⁻¹xsec⁻¹
K _{ass, A1OPSA1} = 1.5x0.4x10⁸M⁻¹xsec⁻¹
K _{ass, A1OPSA1} = 0.6x10⁸M⁻¹xsec⁻¹

The following complex formation rates were obtained for the other expression control sequences:

A1OPSCONA1: K _ass = 1.7x10⁸M⁻¹xsec⁻¹
A1pOPSA1: K _ass = 0.6x10⁸M⁻¹xsec⁻¹
OPA1OPSA1: K _ass = 0.6x10⁸M⁻¹xsec⁻¹

These values can have an error of approximately 15%.

Example 4 Determination of the in vivo promoter strengths (induced) of the expression control sequences A. Principle

The in vivo promoter strengths of the expression control sequences were determined relative to the promoter for the β-lactamase gene (P _bla ) as an internal standard according to the method described by Deuschle et al. (supra) described method determined. To do this pDS3 derivatives with the appropriate expression control sequences were transformed into E. coli M15 cells which contained the plasmid pDMI, 1. The cultures radioactively labeled RNA synthesized in the presence of IPTG (inductor) for a certain period of time were then isolated from cultures of these transformants and then hybridized in separate batches to the following excess single-stranded DNA samples: 1) M13mp9dhfr-DNA, 2) M13mp9bla -DNA and 3) M13mp9-DNA as control. After RNase treatment to break down non-hybridized RNA, the batches were filtered through nitrocellulose filters. Since RNA / DNA hybrids are retained on nitrocellulose, the filter-bound radioactivity is a measure of the amount of RNA present in the individual batches, which is complementary to the DNA samples used. After deducting the radioactivity bound in the control (single-stranded M13mp9-DNA), the ratio of radioactivity bound by single-stranded M13mp9dhfr-DNA (the corresponding RNA was synthesized under the control of the expression control sequence to be measured) to radioactivity bound by single-stranded M13mp9bla-DNA (this RNA was synthesized under control of P _bla ). After a necessary correction, this ratio indicates the promoter strength of the measured expression control sequence in P _bla units.

B. Preparation of single-stranded M13mp9, M13mp9dhfr and M13mp9bla phage DNAs

To produce the phage M13mp9dhfr, the BamHI-HindIII fragment of the plasmid pDS1, t _o 1⁺, containing the dhfr gene in DNA of the phage M13mp9 (Pharmacia Sweden; cut with BamHI and HindIII) was prepared by known methods (Maniatis et al., Supra ) integrated. The phage M13mp9bla was produced in an analogous manner, the EcoRI-PstI Fragment from the plasmid pDS1, t _o 1⁺ with parts of the bla gene in DNA of phage M13mp9 (cut with EcoRI and PstI) was integrated. Thereafter, single-stranded M13mp9-DNA, M13mp9dhfr-DNA and M13mp9bla-DNA were prepared in an analogous manner as described in Example 3 B.3 for the production of single-stranded M13mp8-DNA.

C. Determination of the in vivo promoter strengths (induced)

The determination of the promoter strengths according to that of Deuschle et al. (supra) described procedure was carried out as follows:

1) Production of 3 H-RNA labeled in vivo

E. coli M15 cells, containing the plasmid pDMI, 1, and a pDS3 derivative with one of the various expression control sequences (see Example 2), were stored in 20% glycerol at -20 ° C. 10 ml of LB medium containing 100 μg / ml ampicillin and 25 μg / ml kanamycin were inoculated with an above-mentioned stock culture and grown overnight at 37 ° C. in a shaking incubator (180 rpm). 0.1 ml of this overnight culture was prewarmed in 25 ml of M9 minimal medium (JH Miller, supra), containing 5% casein hydrolyzate, 0.1% bactotrypton, 0.05% yeast extract, 0.05% NaCl, 0.5% glycerol, lmM IPTG, 100 µg / ml ampicillin in 25 ml preheated to 37 ° C and 25 µg / ml kanamycin, diluted. The cells were grown at 37 ° C. in a shaking incubator (250 rpm) to an optical density of OD₆₀₀ = 0.6. 0.5mCi 5,6-3 H-uridine (40-60mCi / mmol, 1mCi / ml aqueous solution; Amersham, Braunschweig, FRG) was added to 10 ml of this culture. After 45 seconds, the culture was quickly cooled to 0 ° C. using liquid nitrogen before the cells were centrifuged off and then resuspended in TES buffer (20 mM Tris-HCl, pH 8.0, 10 mM EDTA, 100 mM NaCl, 1% SDS) were commuted. After incubation for 3 minutes at 95 ° C., the resulting mixture of lysed cells was analyzed according to the method described by Glisin et al. (Biochemistry 13 , 2633-2637 [1974]) centrifuged (CsCl gradient centrifugation, 150,000 × g, 16 hours, 20 ° C.). After removing the supernatant, the centrifuge tubes were cut 0.8 cm above the bottom using a heated scalpel. The RNA in the lower part of the tube was dissolved with 2 x 80 μl TE buffer containing 0.2% SDS and then precipitated with ethanol in the presence of 3M sodium acetate. The precipitate was washed with 80% ethanol, dried in vacuo and then dissolved in hybridization buffer (see below). In general, 200-300 µg RNA with a specific activity of 1-3 x 10⁵ cpm (Cerenkow) / µg RNA were obtained from 10 ml culture.

2) Hybridization of the RNA to an excess of single-stranded DNA

All hybridizations were carried out at 42 ° C. in 20 μl hybridization buffer (50% formamide, 300 mM NaCl, 20 mM Tris-HCL, pH 8.0, 0.5 mM EDTA) for 2 hours. In a typical experiment, 10 µl of the in vivo ³H-RNA (∼5 x 10⁵ cpm) with 10 µl single-stranded M13mp9-DNA (0.2 pMole, control), M13mp9dhfr-DNA (0.2 pMole), or M13mp9bla-DNA (0.2 pMole) mixed, incubated for 3 minutes at 65 ° C and then kept at 42 ° C for 2 hours.

3) Quantification of the hybridized RNA

The hybridization batches were diluted ten times with 2 x SSC buffer and filtered through nitrocellulose filters (0.45 µm BA85 filter, minifold system, Schleicher and Schull, FRG). The capacity of the filters used for single-stranded M13mp9 DNA was approximately 6 pmoles / cm². The filters were washed with 2 ml of 2 x SSC buffer, for 30 Baked in vacuo at 80 ° C for minutes and then incubated for 1 hour at 42 ° C in 100 ml of 2 x SSC buffer containing 50 µg / ml RNase A. The filters were then washed three times with 100 ml of 2 × SSC buffer for 10 minutes at 42 ° C. After the filters had dried, the radioactivity retained on them was counted in universal liquid scintillator (NEN). The ratio of dhfr- to bla-specific RNA was calculated taking into account the number of uridines within the different RNAs. Since the single-stranded DNA inserts specific for dhfr and bla encode 169 or 148 uridines (148/169 = 0.87), the in vivo promoter strength S of a desired expression control sequence in P _bla units results according to the formula:

S = 0.87x (cpm _dhfr -cpm _control ) / (cpm _bla -cpm _control ).

D. In vivo promoter strengths of the expression control sequences

For each of the different expression control sequences, the determination of the in vivo promoter strength, as described in section C, was carried out at least three times, the synthesized, radioactively labeled RNA being determined twice in each case. For the expression control sequence A1OPSA1, for example, the following values were obtained when determining the synthesized RNA: Measurement no. M13mp9 DNA Single-stranded M13mp9dhrf DNA M13mp9bla DNA 1 38 33 581 839 2nd 22 36 099 831

This resulted in the following values for the in vivo promoter strengths:

S₁ - 0.87x (33581-38) / (839-38) = 36.4 P _bla units
S₂ - 0.87x (36099-22) / (831-22) = 38.8 P _bla units

An in vivo promoter strength of 37.6 P _bla units was calculated as the mean.

Averaged over a total of three determinations, a promoter strength of 38.1 ± 3.4 P _bla units was calculated for the expression control sequence A1OPSA1. Table 3 lists the promoter strengths determined as described above for all expression control sequences tested. Table 3 Expression control sequence in vivo promoter strength, induced [P _bla units] lacOP29 5.5 ± 1.0 tacOP29 17.6 ± 1.8 N25 26.2 ± 2.0 N25 * / 0 8.0 ± 1.5 N25OP29 7.7 ± 1.3 N25OPSN25OP29 9.3 ± 1.2 A1 66.1 ± 2.5 A1OPSA1 38.1 ± 3.4 A1OPSCONA1 16.8 ± 2.0 A1pOPSA1 25.1 ± 1.3 OPA1OPSA1 38.1 ± 3.4 A10PSA10P29 31.5 ± 3.0 A10PSA10P21 32.9 ± 5.0

Example 5 Determination of the repression factor of the different expression control sequences A. Principle

The repression factors, i.e. the ratio of the in vivo promoter strengths under induced and repressed conditions for the individual expression control sequences were determined as follows:

a) Expression control sequences with a repression factor of less than 100

The expression control sequences tacOP29, N25 * / 0, N25OP29, OPUA1 and OPUA1CON initiate sufficient amounts of RNA in vivo under repressed conditions (excess repressor, no inducer) so that these could be determined directly. The repression factors were then calculated using the values for the in vivo promoter strength (induced) determined in Example 4.

b) expression control sequences with a repression factor of greater than 100

For the expression control sequences lacOP29, N25OPSN25OP29, A1OPSA1, A10PSA10P21, A10PSA10P29, A1OPSCONA1, A1pOPSA1 and OPA1OPSA1, the in vivo promoter strength was determined indirectly under repressed conditions. For this purpose, the amount of β-galactosidase that was produced in a suitable system under repressed conditions was first determined for the individual elements by means of an enzymatic test. The so The values obtained were then converted into P _bla units using a correction factor. The repression factors were then calculated using the values determined in Example 4.

B. Direct determination of in vivo promoter strengths (P _blah Units) of the expression control sequences under repressed conditions

The in vivo promoter strengths under repressed conditions were determined as described in Example 4, but without the addition of IPTG. The values obtained are summarized in Table 4: Table 4 Expression control sequence Promoter strengths, repressed [P _bla units] tacOP29 0.36 ± 0.02 N25OP29 1.5 ± 0.1 N25 * / 0 1.5 ± 0.1 OPUA1 2.7 ± 0.5 OPUA1CON 1.7 ± 0.6

C. Indirect determination of the in vivo promoter strengths of the expression control sequences under repressed conditions 1. Determination of the β-galactosidase units

The pML3 derivatives with the expression control sequences lacOP29, tacOP29, N25OP29, N25OPSN25OP29, A1OPSA1, A10PSA10P21, A10PSA10P29, A1OPSCONA1, A1pOPSA1, OPA1OPSA1, OPUA1 and OPUA1Ci were first described in the methods described in E coli M15 cells transformed. The resulting transformed E. coli M15 cells with the corresponding plasmids were then grown to the logarithmic phase and the amount of β-galactosidase was determined according to the instructions of J.H. Miller ("Experiments in Molecular Genetics", Cold Spring Harbor, New York, 1972). Such a determination is described below using the example of the expression control sequence N25OP29.

10 ml of supplemented minimal medium (JH Miller, supra) were inoculated with 0.05 ml of an overnight culture of transformed E. coli M15 cells containing plasmid pML3 / N25OP29, and incubated at 37 ° C. in a shaking incubator (200 rpm). After reaching an OD₆₀₀ (optical density at a wavelength of 600 nm) of 0.55, the culture was placed on ice for 20 minutes. The optical density was then determined again. A value of OD₆₀₀ = 0.607 was obtained. 0.1 ml of the culture was then diluted to a final volume of 1 ml with Z buffer (JH Miller, supra). This sample was treated with a control (1 ml Z buffer) as follows:
Addition of 60 µl chloroform and 30 µl 0.1% SDS (cell disruption);
Mix the sample for 10 seconds using vortex;
Incubate the sample for 5 minutes at 28 ° C;
Addition of 200 ul ONPG (JH Miller, supra);
Mixing the sample by vortex;
Incubate the sample at 28 ° C until yellowing became visible;
Addition of 250µl 2M Na₂CO₃ (the time between the addition of ONPG and Na₂CO₃ was 1.5 minutes);
Mixing the sample by vortex;
Centrifugation of the cell debris (Eppendorf table centrifuge, 13000 rpm, 5 minutes); and
Determination of the absorbance at 420 nm against the control.

A value of ΔE₄₂₀ = 0.737 was obtained. According to the formula described in the aforementioned textbook by JH Miller:

β-galactosidase units = 1000 x ΔE₄₂₀ / OD₆₀₀xVxt

where ΔE₄₂₀ is the E₄₂₀ measured value of the reaction mixture against the control (0.737), OD₆₀₀ is the cell density of the culture sample used (0.607), t is the reaction time (1.5 minutes) and V is the volume of the culture used (0.1 ml),
8094 β-galactosidase units were calculated. Averaged over 8 experiments, 8160 ± 450 β-galactosidase units were obtained.

Table 5 summarizes the values obtained for all measured expression control sequences, with at least 4 measurements being carried out for each expression control sequence. Table 5 Expression control sequence β-galactosidase units lacOP29 30 ± 5 tacOP29 1510 ± 170 N25OP29 8160 ± 450 N25OPSN25OP29 99 ± 6 A1OPSA1 110 ± 2 A1OPSCONA1 220 ± 12 A1pOPSA1 58 ± 3 OPA1OPSA1 56 ± 1 OPUA1 13700 ± 3000 OPUA1CON 8500 ± 2000 A10PSA10P29 23 ± 3 A10PSA10P21 15 ± 3

2. Determination of the factor for converting β-galactosidase units into P. _blah -Units

For the expression control sequences tacOP29, N25OP29, OPUA1 and OPUA1CON, the in vitro promoter strengths could be determined both directly (P _bla units, Table 4) and indirectly (β-galactosidase units, Table 5) under repressed conditions. The values obtained are plotted against each other in Fig. 21. The slope of the regression line is a measure for the conversion of the β-galactosidase units into P _bla units. A value of 5000 β-galactosidase units / P _bla unit was determined.

Conversion of β-galactosidase units to P _blah Units

Using the conversion factor 5000 β-galactosidase units / P _bla unit, the P _bla units were calculated for the corresponding expression control sequences from the measured β-galactosidase units (Table 5) (Table 6). Table 6 Expression control sequence β-galactosidase units P _bla units lacOP29 30 ± 5 0.006 ± 0.001 N25OPSN25OP29 99 ± 6 0.02 ± 0.003 A1OPSA1 110 ± 2 0.022 ± 0.001 A1OPSCONA1 220 ± 12 0.044 ± 0.003 A1pOPSA1 58 ± 3 0.012 ± 0.001 OPA1OPSA1 56 ± 1 0.011 ± 0.001 A10PSA10P29 23 ± 3 0.0046 ± 0.0006 A10PSA10P21 15 ± 3 0.003 ± 0.0006

D. Calculation of repression factors

To calculate the repression factors (P _bla units induced / P _bla units, repressed) for the individual expression control sequences, the values listed in Tables 3, 4 and 6 were used (Table 7). Table 7 in vivo promoter strengths Expression control sequence P _bla units P _bla units, repressed Repression factor lacOP29 5.5 ± 1.0 0.006 ± 0.001 920 ± 230 tacOP29 17.6 ± 1.8 0.36 ± 0.02 ^a 49 ± 6 N25 * / 0 8.0 ± 1.5 1.5 ± 0.1 ^a 5.3 ± 2 N25OP29 7.7 ± 1.3 1.5 ± 0.1 ^a 5.1 ± 2 N25OPSN25OP29 9.3 ± 1.2 0.02 ± 0.003 465 ± 92 A1OPSA1 38.1 ± 3.4 0.022 ± 0.001 1730 ± 170 A1OPSCONA1 16.8 ± 2.0 0.044 ± 0.003 380 ± 50 A1pOPSA1 25.1 ± 1.3 0.012 ± 0.001 2090 ± 240 OPA1OPSA1 38.1 ± 3.4 0.011 ± 0.001 3460 ± 460 A10PSA10P29 31.5 ± 3.0 0.0046 ± 0.0006 6850 ± 610 A10PSA10P21 32.9 ± 5.0 0.003 ± 0.0006 10970 ± 2000 ^a direct determination of the in vivo promoter strength

E. Summary of the properties characterizing the individual expression control sequences

The following table 8 summarizes the values obtained for the individual expression control sequences for the in vitro complexation rate, the in vivo promoter strength and the repression factor; Table 8 Expression control sequence in vitro complex formation rate (K _ass ) [10⁸M⁻¹ x sec⁻¹] in vivo promoter strengths Repression factor repressed [P _bla units] induced [P _bla units] lacOP29 0.02 ^a 0.006 ± 0.001 5.5 ± 1.0 920 ± 230 tacOP29 0.85 0.36 ± 0.02 17.6 ± 1.8 49 ± 6 N25 2.9 - 26.2 ± 2.0 - N25 * / 0 2.9 1.5 ± 0.1 8.0 ± 1.5 5.3 ± 2 N25OP29 2.9 1.5 ± 0.1 7.7 ± 1.3 5.1 ± 2 N25OPSN25OP29 2.9 0.02 ± 0.003 9.3 ± 1.2 465 ± 92 A1 1.5 - 66.1 ± 2.5 - * A1OPSA1 0.6 0.022 ± 0.001 38.1 ± 3.4 1730 ± 170 A1OPSCONA1 1.7 0.044 ± 0.003 16.8 ± 2.0 380 ± 50 * A1pOPSA1 0.6 0.012 ± 0.001 25.1 ± 1.3 2090 ± 240 * OPA1OPSA1 0.6 0.011 ± 0.001 38.1 ± 3.4 3460 ± 460 * A1OPSA1OP29 nb 0.0046 ± 0.0006 31.5 ± 3.0 6850 ± 610 * A1OPSA1OP21 nb 0.003 ± 0.0006 32.9 ± 5.0 10970 ± 2000 ^a McClure et al. ("Promoters, Structure and Function", ed. Rodriguez and Chamberlin, Praeger, pages 111-120 [1982]) * expression control sequences according to the invention, nb = not determined.

Claims (32)

1. An expression control sequence characterized by the combination of promoter sequences with low signal strength and high in vivo promoter strength with operator / repressor systems with a high complex formation rate. 2. An expression control sequence according to claim 1, characterized in that the promoter sequence with low signal strength and high in vivo promoter strength has a signal strength of approximately 1 · 10⁶-1.5 · 10⁸M⁻¹ · sec⁻¹ and an in vivo promoter strength of 10-100 P _bla Units. 3. An expression control sequence according to claim 1 or 2, characterized in that the promoter sequence with low signal strength and high in vivo promoter strength has a signal strength of about 6 · 10⁷M⁻¹ · sec⁻¹ and an in vivo promoter strength of more than 20 P _bla units having. 4. An expression control sequence according to any one of claims 1-3, characterized in that the operator / repressor system with high complex formation rate has a complex formation rate of about 1 · 10 ·-1 · 10¹¹M⁻¹ · sec⁻¹. 5. An expression control sequence according to claim 4, characterized in that the operator / repressor system with a high complex formation rate has a complex formation rate of 2 · 10⁹M⁻¹ · sec⁻¹. 6. An expression control sequence according to any one of claims 1-5, which is obtained by combining promoter sequences with operator / repressor systems from gram-negative organisms. 7. An expression control sequence according to claim 6, which is obtained by combining promoter sequences from T-Coliphagen with the lac operator / repressor system. 8. An expression control sequence according to claim 7, which is obtained by combining the T7AI promoter with the lac operator / repressor system. 9. An expression control sequence according to claim 8 with the functional parts of the nucleotide sequence

10. An expression control sequence according to claim 8, with the functional parts of the nucleotide sequence

11. An expression control sequence according to claim 8 with the functional parts of the nucleotide sequence

12. An expression control sequence according to claim 8 with the functional parts of the nucleotide sequence

13. An expression control sequence according to claim 8 with the functional parts of the nucleotide sequence

14. An expression vector. which contains an expression control sequence according to any one of claims 1-13. 15. An expression vector according to claim 14, which can replicate in gram-negative and / or gram-positive bacteria. 16. An expression vector according to claim 15, which can replicate in E. coli. 17. An expression vector according to claim 15, which can replicate in B. subtilis. 18. An expression vector according to claim 15, which can replicate in Salmonella typhimurium. 19. A bacterium containing an expression vector according to claims 14-18. 20. A bacterium according to claim 19 which is an E. coli cell. 21. A bacterium according to claim 20 which is an E. coli M15 cell. 22. A bacterium according to claim 19 which is a B. subtilis cell. 23. A bacterium according to claim 19 which is a Salmonella typhimurium cell. 24. A bacterium containing an expression control sequence according to any one of claims 1-13 in the chromosome. 25. A bacterium according to claim 24 which is an E. coli cell. 26. A bacterium according to claim 24 which is a B. subtilis cell. 27. A bacterium according to claim 24 which is a Salmonella typhimurium cell. 28. A process for the production of a pro- or eukaryotic protein, characterized. that a bacterium is transformed with an expression vector in which the DNA sequence coding for said protein is operably linked to an expression control sequence according to any one of claims 1-13, then cultivated under suitable conditions and the said protein is isolated and, if desired, purified . 29. The method according to claim 28, wherein the bacterium is an E. coli, a Salmonella typhimurium or B. subtilis cell. 30. A process for the production of a pro- or eukaryotic protein, characterized in that one with the expression control sequence according to any one of claims 1-13 incorporates an operably linked coding DNA sequence for said protein into the chromosome of a bacterium, this bacterium is then cultivated under suitable conditions and the said protein is isolated and, if desired, purified. 31. The method according to claim 30, wherein the bacterium is an E. coli, a Salmonella typhimurium or B. subtilis cell. 32. Use of an expression control sequence according to any one of claims 1-13 for the expression of pro- and eukaryotic proteins.

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